Method for producing C2 oxygenates by fermentation using high oxidation state sulfur

ABSTRACT

The invention relates to improvements in the production of ethanol and acetate by microbial fermentation, particularly to production of alcohols by microbial fermentation of a substrate comprising CO and the addition of an inorganic sulfur additive. It more particularly relates to the provision of an inorganic organic sulfur source to a fermentation system such that one or more microorganisms convert a substrate comprising CO to ethanol. In one aspect the invention uses a sulfur additive comprising inorganic sulfur compounds having a +2 to a +4 sulfur oxidation state that produces sulfur oxoanions and hydrosulfur oxoanions in an aqueous fermentation medium.

FIELD OF THE INVENTION

This invention relates to method for the production of ethanol and/oracetate from substrates of carbon monoxide and/or carbon dioxide andhydrogen by anaerobic fermentation using carboxydotrophicmicroorganisms. More particularly, the invention pertains to usingspecific sources of inorganic sulfur to supply the biological sulfurneeds of the microorganisms in a manner that is more cost effective andbeneficial to such production of ethanol and acetate.

BACKGROUND

Biofuels production for use as liquid motor fuels or for blending withconventional gasoline or diesel motor fuels is increasing worldwide.Such biofuels include, for example, ethanol and n-butanol. One of themajor drivers for biofuels is their derivation from renewable resourcesby fermentation and bioprocess technology. Conventionally, biofuels aremade from readily fermentable carbohydrates such as sugars and starches.Since these feed sources compete with the human food supply much recentwork has focused on alternative feed sources for biofuels and otherchemicals.

One alternate source of feeds is lignocellulosic feedstocks such asforest residues, trees from plantations, straws, grasses and otheragricultural residues. However, the very heterogeneous nature oflignocellulosic materials that enables them to provide the mechanicalsupport structure of the plants and trees makes them inherentlyrecalcitrant to bioconversion. Also, these materials predominantlycontain three separate classes of components as building blocks:cellulose (C6 sugar polymers), hemicellulose (various C5 and C6 sugarpolymers), and lignin (aromatic and ether linked hetero polymers). Thenother well-known technologies can convert the lignocellulosic biomassfeed to syngas (also known as synthesis gas, primarily a mix of CO, H₂and CO₂ with other components such as CH₄, N₂, NH₃, H₂S and other tracegases) This syngas is then fermented with anaerobic microorganisms toproduce biofuels such as ethanol, n-butanol or chemicals such as aceticacid, butyric acid and the like. U.S. Pat. No. 7,285,402, the teachingsof which are incorporated by reference herein, discloses methods forconverting carbon monoxide, carbon dioxide, and hydrogen to acetic acidand ethanol by fermentation using anaerobic bacteria.

Anaerobic fermentations to produce biofuels and other chemicals canutilize any gaseous substrates that provide a carbon monoxide and/orcarbon dioxide and hydrogen from a variety of sources. For example, USPatent Publication 2011/0300593 discloses sources such as steel milloff-gas as source of carbon monoxide and the teachings of which areincorporated by reference herein. Many other sources of substrates areavailable. For example, syngas can be made from many other carbonaceousfeedstocks such as natural gas, reformed gas, peat, petroleum coke,coal, solid waste and land fill gas.

Ethanol can be produced from CO, CO₂ and H₂ using a variety of anaerobicbacteria, in particular such as those from the genus Clostridium. Forexample, various strains of bacteria that produce ethanol from gases aredescribed and include Clostridium ljungdahlii, Clostridiumautoethanogenum and Clostridium coskatii all of which are describedfurther herein.

The production of ethanol and other products by the anaerobicmicroorganisms is influenced by many operating conditions within thefermentation zone. (see U.S. Pat. Nos. 5,173,429, 5,593,886, and6,368,819, WO 98/00558 and WO 02/08438) Two primary conditions affectingthe microorganism performance are the pH and oxidation-reductionpotential (ORP) of the fermentation zone. WO2009/022925 discloses theeffect of pH and ORP in the conversion of the gaseous substrates toproducts.

Microorganisms used in metabolic processes require nutrients andmicronutrients and the particular supply of the nutrients can haveprofound effects on the growth and sustainability of the microorganisms.In fact these nutrients may be required to enable the microorganism touse carbon monoxide as its source of energy. For example, themicroorganism may require the presence of metal co-factors for themetabolic functions of carbon monoxide dehydrogenase (CODH), andacetyl-CoA synthase (ACS). It is important that all of the requirednutrients are provided in the proper amount and a bioavailable form.

Another of the required nutrients is a source of reduced sulfur, usuallyin the form of an organic sulfide such as cysteine. The cysteineprovides a sulfur source necessary to support enzymatic processesoccurring in a microbial culture. It is well known that microorganismsrequire sulfur in their enzymatic processes. In fact, the electrontransfer mediator, ferredoxin, as well as Wood-Ljungdahl pathway enzymesacetyl-CoA synthase, and carbon monoxide dehydrogenase contain sulfur.Therefore, it is important to add sulfur in a bioavailable form and insufficient supply to avoid inhibiting the growth or production ofproduct by the microorganism.

As an alternative to cysteine, hydrogen sulfide has been found in manyinstances to be a source of the reduced sulfur needed for the metabolicprocesses of the microorganisms. Sulfur sources such as sulfide exist inequilibrium with hydrogen sulfide in typical fermentation media.Although hydrogen sulfide is less expensive than cysteine, it is toxicand thus requires special handling and is particularly dangerous in pureform. Supplying sulfur in the form of a sulfide salt such as sodiumsulfide still results in a hydrogen sulfide concentration in thefermenter that may decrease over time due to evaporation. Moreover,hydrogen sulfide has a limited solubility in the media. Hydrogen sulfidemay become highly volatile under the certain conditions that may bedesired in the fermentation zone thereby exacerbating its use as asulfur source. As a result, of the limited solubility of hydrogensulfide in water the concentration of sulfur in solution can besignificantly reduced by the conditions within fermentation medium.Accordingly, identification of improved or alternative sulfur sourcesfor the microorganism in alcohol production required in fermentationsystems using carbon monoxide or hydrogen and carbon dioxide gases as afeedstock would aid in achieving high alcohol production rates and lowprocess operating costs.

Accordingly, the methods for producing ethanol or acetate from anaerobicfermentations would benefit from the discovery of sulfur compounds thatcan inexpensively provide the biological sulfur needs of themicroorganisms under favorable fermentation conditions while alsoeliminating the disadvantages of hydrogen sulfide as a sulfur source.

US Patent Publication 20110300593 discloses the use of alternate sulfursources. This document only specifically describes polysulfide,polysulfides, elemental sulfur and colloidal sulfur as the inorganicsulfur compounds for use as an alternative sulfur source. Moreimportantly, the document describes the use of these specific sulfurcompounds as means of providing hydrogen sulfide as the active speciesin the fermentation medium. As a result, this reference does notidentify a sulfur additive that will provide a biologically availableform of sulfur other than hydrogen sulfide and therefore does notovercome the high volatility problems of hydrogen sulfide.

Methods are sought to enhance the economics of syngas fermentation toproduce C2 oxygenated organic compounds where the sulfur nutrient can beeffectively and inexpensively supplied by the processes at an “asneeded” rate.

SUMMARY OF THE INVENTION

This invention provides methods for the bioconversion of syngas to a C2oxygenated organic compounds such as ethanol and acetate where thesupply of the sulfur nutrient is provided by a sulfur additive thatcontributes sulfur oxyanions or hydrosulfur oxyanions to liquid nutrientmedium in a form that meets the metabolic needs of the microorganismwhile facilitating operation of the fermentation zone at pH that willinhibit the growth of contaminating microorganisms. It was surprisinglydiscovered that such compounds can supply the nutritional sulfur needsof the carboxydotrophic microorganisms in the production of ethanol andacetate from substrates of CO, CO₂ and H₂. This class of compoundsincludes the bisulfites that are routinely used as antimicrobial agentsin various food products. Ordinarily one would have expected suchcompounds to interfere with the growth and viability of microorganisms.Thus, it was surprising to discover that such compounds can be usedbeneficially in providing the sulfur species that will satisfy themetabolic requirements of the microorganisms while avoiding thevolatility problems of hydrogen sulfide at lower pH conditions in thefermentation zone.

In a broad aspect this invention provides a method for the production ofC2 oxygenates using ethanol producing carboxydotrophic microorganismswhile supplying sulfur to the fermentation medium in a bioavailable andhighly soluble form. Maintaining a pH below 5.3 and more preferablybelow 5.1 is highly beneficial in inhibiting the growth of contaminatingmicroorganisms in the fermentation zone. Common contaminants that canalter or disrupt the fermentation process are microorganisms thatproduce butyrates and can reduce the selectivity of the process for moredesired ethanol products.

In supplying the higher oxidation state sulfur the invention uses themicroorganism as the means to reduce the oxidation state of the sulfurto the usable form. It is proposed that in this manner the microorganismproduces the hydrosulfide (HS⁻) that it assimilates within the cellwhere it is used. This reduces the presence HS⁻ in the liquid nutrientmedium and helps to minimize any equilibrium concentration of hydrogensulfide that would contribute to hydrogen sulfide emissions from thefermentation.

In particular embodiments, this invention is a method for producing C2oxygenates by anaerobic fermentation with an ethanol-producingcarboxydotrophic microorganism. The method comprises providing a sulfuradditive comprising an S(II) to S(IV) inorganic sulfur compound thatproduces sulfur anions containing oxygen and/or oxygen and hydrogenatoms in an aqueous fermentation medium, contacting a microbial cultureof the microorganism with a substrate comprising carbon monoxide,maintaining the microbial culture in the fermentation medium containingthe sulfur additive and having a pH of less than 5.3, and recovering oneor more C2 oxygenates from the fermentation medium. In a furtherembodiment the sulfur additive comprises an S(III) to S(IV) inorganicsulfur compound.

In a particular embodiment, the sulfur additive is sulfurous acid,bisulfite, metabisulfite, dithionite, thiosulfate, or a combinationthereof. In a preferred embodiment, the sulfur additive is sulfurousacid, bisulfite, metabisulfite or combination thereof. In otherembodiments the sulfur additive may include sodium bisulfite, sodiummetabisulfite.

In a particular embodiment, the sulfur additive is present in thefermentation medium at a concentration of at least 0.1, typically in arange of 0.1 to 10 and preferably in a range of 0.5 to 2 mmol sulfur pergram dry cell weight of microorganism.

In a particular embodiment, the contacting of the microorganisms withthe sulfur additive takes place in the presence of an added organicsulfur source and the concentration of the organic sulfur in thefermentation medium is reduced over time. In a preferred embodiment, theconcentration of the organic sulfur in the fermentation medium is lessthan 0.3 mmol organic sulfur per gram dry cell weight of microorganism,and more preferably less than 0.1 mmol organic sulfur per gram dry cellweight of microorganism.

In another aspect, the invention provides a method of eliminatingcysteine as an amino acid and sulfur source for addition to the liquidnutrient media of the fermentation. In a particular embodiment of thisinvention, the concentration of cysteine in the fermentation medium maybe significantly decreased and preferably decreased to a concentrationof less than 0.3 mmol per gram dry cell weight of microorganism. In aparticular embodiment, cysteine addition to the fermentation medium maybe eliminated completely.

In another aspect of the invention, the pH of the fermentation medium iskept in a range of 4.3 to 5.1, and preferably below 4.9.

In another aspect of the invention, the method includes a chelatingagent in the fermentation medium. In a preferred embodiment, theinvention uses ethylenediaminetetraacetic acid,diethylenetriaminepentaaceteic acid, nitrilotriacetic acid, sodiumcitrate, and mixtures thereof as a chelating agent.

Generally, ethanol is the preferred metabolite for production by themicroorganisms. In most cases the other metabolites will be producedtogether with acetate in the fermentation of the substrate. It has beenfound that the methods of the invention can improve the alcohol to acidproduct ratio. Product ratios in the range of ethanol to acetate ofgreater than 5:1 and greater than 10:1 have been found along with higherratios of 20:1 or more favoring alcohol over acetate.

One application of the invention is in the case where an organic sulfursource such as cysteine is used in first vessel to grow the microbialculture and then the microbial culture is transferred to a secondvessel. The first vessel may also contain the sulfur additive ifdesired. The contacting of the microbial culture from the first vesselwith the substrate may then take place in the separate second vessel.The second vessel may maintain the fermentation with little or nocysteine. In particular embodiments of this invention, the secondfermentation vessel will be a larger volume fermentation vessel aspracticed in seed train operations to passage the microbial culture upto commercial scale volumes. Such transfers of the microbial culture cancontinue to a third fermentation zone and to additional fermentationzones of higher volume. In a preferred embodiment, the concentration oforganic sulfur such as cysteine will decrease as the volume of thefermentation zone increases. In a further embodiment the addition ofcysteine to the fermentation zone will only continue in fermentationzones having a volume of less than 40,000 liters and more preferablyless than 4,000 liters.

Accordingly in another embodiment, the invention is a method forreducing cysteine in the production of ethanol or acetate by anaerobicfermentation. The method comprises contacting a microbial culture of acarboxydotrophic microorganism with a substrate comprising carbonmonoxide and growing the microbial culture under anaerobic conditions ina first fermentation zone containing cysteine at a first concentrationof cysteine to produce a cysteine grown microbial culture in a firstfermentation zone. Some or all of the cysteine grown microbial cultureis transferred to a second fermentation zone containing a sulfuradditive comprising an inorganic sulfur compound having a +2 to a +4oxidation state (S(II)-S(IV)) that produces anions in the secondfermentation zone consisting of sulfur atoms combined with oxygen and/orhydrogen atoms. A substrate comprising (i) carbon monoxide, (ii) carbondioxide and hydrogen, or (iii) mixtures of (i) and (ii) is added to thesecond fermentation zone to convert the substrate to C2 oxygenates. Oneor more C2 oxygenates are recovered from the second fermentation zone.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 through 22 are plots of fermentation data showing gas uptake andproduct production from a series of continuation fermentation runs usingthe sulfur additive of this invention.

DEFINITIONS

“Bioreactor” means any fermentation apparatus having at least one vesselfor retaining microorganisms in the presence of a fermentation mediumwith the term vessel referring to any containment device includingpipes, columns, membranes, screen and other like devices that can bearranged as continuous or batch reactors and may comprise a BubbleColumn Bioreactor (BCBR), Continuous Stirred Tank Reactor (CSTR), anImmobilized Cell Reactor (ICR) such as a Membrane Supported Bioreactor(MSBR), jet loop reactors, trickle bed reactors, pipe reactors or anyother device that provides the gas liquid contacting of themicroorganisms with the substrate. The fermentation reactors used inthis invention may be of any suitable design; however, preferably thedesign and operation provides for a high conversion of carbon monoxideand hydrogen to oxygenated organic compound.

“C2 oxygenate” means any molecule containing 2 carbon atoms, hydrogenatoms and at least one oxygen atom. Preferably, the C2 oxygenates willcomprise ethanol, acetic acid and acetate anions.

“Microbial culture” means a mixture of microorganisms together with aliquid fermentation medium wherein the microorganisms are present in theform of a slurry, a suspension or fixed colonies. Microbial cultureincludes microorganisms suspended on moving support media such tubesrings or biochips or fixed on support media such as membranes or otherporous or semi-porous media. The term microbial culture includescultures made of a single strain of microorganism or multiple strains ofmicroorganisms.

“Fermentation medium” means an aqueous liquid that contacts themicroorganism and contains nutrients such as vitamins, minerals, aminoacids, etc. that the microorganism needs for maintaining its biologicalfunctions as well as metabolic products produced by the microorganismsthat are recovered from the fermentation medium. The fermentation mediummay keep the microorganism suspended in planktonic state, may serve as asuspension medium to retain carrier material upon which themicroorganisms are grown or may perfuse to and from the microbialculture across a support such as a membrane. The fermentation medium maydirectly receive the gaseous substrate and serve as the means forsupplying the substrate to the microorganisms.

“Fermenting” or “fermentation” means any conversion of substrate by amicroorganism whether the microorganism is in a phase of primary growthfor the organism or in a phase of primarily producing metabolites asproduct.

“Mean cell retention time” means for continuous fermentation operationsthe total mass of the cells in the fermenter divided by the rate atwhich the biomass leaves the fermenter.

“Nutrient liquid” means an aqueous liquid that contains one or more ofthe nutrients such as vitamins, mineral, or amino acids needed by themicroorganism to support or improve its biological functions and that isadded to the fermentation medium or directly contacts a microbialculture.

“Organic sulfur” means any organosulfur compound defined as any sulfurderivative having at least one alkyl or aryl group.

The value of the term “redox potential” or “ORP” when quantified hereinis taken as the measurement of such value with reference to an aqueoussolution measured against Ag/Ag—Cl type electrode utilizing a 3.8M KClelectrolyte salt bridge.

“S(II)”, “S(III)”, and “S(IV)” mean a positive oxidation state of two,three, and four respectively of the sulfur atoms in a sulfur compound.

“Substrate(s)” means a gas or gases that provides the primary carbon andenergy source to the microorganism and comprises at least one of carbonmonoxide or a combination of carbon dioxide and hydrogen.

DETAILED DESCRIPTION OF THE INVENTION

This invention uses a unique class of inorganic sulfur compounds toprovide the required sulfur to a fermentation process for the productionof C2 oxygenates from the gaseous substrates that contain one or more ofcarbon monoxide and/or a combination of carbon dioxide and hydrogen. Inmost instances the primary advantage of the process is the eliminationof the need for cysteine from the fermentation zone. In certainembodiments it may be possible to remove cysteine at the initiation ofthe fermentation or cysteine may be reduced and/or completely phased outas the fermentation progresses. This means the cysteine can be reducedto much lower levels than would be needed if cysteine were the primarysource of bioavailable sulfur or may be eliminated from on-goingfermentation operations not only as a sulfur source but also as a directsource of an amino acid. The inorganic sulfur additives of thisinvention have been found to provide sulfur in a highly sustainable formwithin the fermentation zone and to also enable the microorganism tothrive in both growth and production phases so that high titers ofethanol can be achieved from the fermentation section.

An essential component of this invention is the provision of a specifictype of sulfur additive. The sulfur additive that may be used in thisinvention will comprise sulfur compounds having sulfur atoms present ina +2 to +4 oxidation state (S(II)-S(IV)). These additives can be anytype of sulfur compounds that will provide sulfur oxoanions and/orhydrosulfur oxoanions. Particular anionic forms of the additive includesulfite, bisulfite, metabisulfite, sulfur dioxide, dithionite, andthiosulfate. The additive may be derived by the addition of varioussulfur compounds such as sulfurous acid, sodium bisulfite, sodiummetabisulfite, potassium metabisulfite, dithionous acid, sodiumdithionite, sodium thiosulfate

The sulfur additive once dispersed into the aqueous liquid of thefermentation medium will be present in its active anionic form. Theoxoanions of sulfur and hydrosulfur oxoanions that are effective in thisinvention comprise the sulfite form SO₃ ²⁻ and hydrogen sulfite(bisulfite) form HSO₃ ⁻. While not wishing to be bound by any theory,the invention relies on the ability of the microorganisms to transportthese sulfur oxoanions across the membrane of its cellular structure.Once inside the cell the microorganisms reduce the oxyanions to HS⁻. Thereaction sequence likely proceeds: SO₃ ²⁻=>H₂S via sulfite reductase.Then, HS⁻+O-acetylserine=>cysteine via O-acetylserine sulfhydrylase.When exposed to water the sulfur compounds will dissociate intoequilibrium mixtures of the sulfite anion and other positively chargedspecies or neutral molecules. For example, using a sulfurous acidsolution in water as an additive will result in an equilibrium of sulfurdioxide and the bisulfite ion according to the following formula:H₂SO₃+H2O

HSO₃ ⁻+H⁺

With the disassociation constants—

K_(a)=1.38×10⁻²; pK_(a)=1.86.

Similarly, aqueous sulfur dioxide will bind with water and deprotonateaccording to the combined formula and pKa:SO₂+H₂O

HSO₃ ⁻+H⁺

with the disassociation constants—

K_(a)=1.54×10⁻²; pK_(a)=1.81.

The hydrogen sulfite will also exist in equilibrium with the sulfite ionaccording to the formula:HSO₃ ⁻

SO₃ ²⁻+H⁺

and is a weakly acidic species with a pK_(a) of 6.97.

The dithionite anion ([S₂O₄]²⁻), is another sulfur oxoanion that may bederived for use in this invention from dithionous acid and H₂S₂O₄ thatwill upon hydrolysis yield an equilibrium mixture of thiosulfate andbisulfite according to the formula:2S₂O₄ ⁻²+H₂O→S₂O₃ ⁻²+2HSO₃ ⁻¹.

Suitable sulfur additives are available in a wide variety of forms thatwill provide the sulfur additive and the oxoanions of sulfur forassimilation by the microorganisms. Those skilled in the art willappreciate that suitable inorganic salts of sulfur may be used to supplythe sulfur additive of this invention. The usual forms of the additiveswill comprise sodium bisulfite, sodium metabisulfite, sulfurous acid,potassium metabisulfite, potassium bisulfite, or any othercationic-oxoanionic-sulfur combination. It is highly beneficial that thesulfur compound is substantially soluble in the fermentation medium andis non-toxic to the microbial culture at the effective concentration ofthe sulfur additive at any point in the microbial culture.

The invention will maintain the sulfur additive at or above apredetermined level. The sulfur additive can be added in an amount tosupply all or a portion of the sulfur needed to meet the biologicalneeds of the microorganisms. The sulfur additive is typically suppliedin an amount sufficient to provide enough individual atoms of sulfur tomeet the biological needs of the microorganisms. For steady statefermentation conditions supplying all of the sulfur for the biologicalneeds of the microorganism typically requires a sulfur addition rate ofat least 0.25 mmols of the sulfur additive per gram dry cell weight ofthe microorganism for monoatomic sulfur ions with such rate adjusted forions with multiple sulfur atoms. A preferred rate of sulfur addition isin the range of 0.67 to 2 mmols of sulfur per gram dry cell weight ofmicroorganism.

The sulfur additive can comprise a variety of inorganic sulfur sourcesthat may decompose and/or release into the bioavailable sulfur speciesof this invention at different rates depending on variables such as pH,temperature, pressure, etc. The supply of the sulfur additive may needto vary over time in accordance with the decomposition anddisassociation rates of the particular sulfur additive that is in use.Such adjustments in the type and/or amount of sulfur additive supply maybe necessary to support growth and/or productivity of the microbialculture. Therefore, the fermentation may benefit from increasing thedesirable sulfur species by supplying greater amounts of the sulfuradditive or varying the type of sulfur additive at different times inthe various stages of the fermentation.

In particular embodiments, the one or more inorganic sulfur compound isa solution of bisulfite compound such as sodium bisulfite obtained inpreferred form as 5 molar bisulfite solution and diluted to provide astock solution at a preferred concentration from of about 1.2 mM. Theconcentration of bisulfite in solution will depend on various factorsincluding pH and solubility.

In another embodiment, it has been discovered that carboxydotrophicmicroorganisms can survive in growth media that does not include anysupplemental amino acids, including cysteine, and whose source of sulfuris sulfurous acid (H₂SO₃). The sulfur atoms of sulfurous acid andmetabisulfite exist in the +4 oxidation state. It is known thatmetabisulfite, sulfurous acid, sulfite, sulfur dioxide, and bisulfiteall exist in equilibrium in water. T. Fazio and C. R. Warner. 1990. AReview of Sulphites in Foods: analytical methodology and findings. FoodAddit. Contam. 7:433-454.

In a particular form of this embodiment, a microbial culture may beinitiated with cysteine as a sulfur additive and subsequently receivereduced concentrations of cysteine (along with proportional increases inthe sulfur additive) while experiencing little sustained change inhydrogen uptake. Agitation and gas flow may be manipulated to controlgas mass transfer where desired. Satisfactory results can be obtainedeven where the nutrient liquid is fully free of metabisulfite andcysteine for periods of over 800 hours and sulfurous acid is left as theonly sulfur source supplied by the nutrient liquid for assimilation bythe microorganisms. (Sulfate anions are present in the media, but thetypical microorganisms for use in this invention do not utilize sulfatein either an assimilatory or dissimilatory fashion.) In some casesserine addition may be useful as well.

In some cases, excess addition of S(IV) compounds may result in reducedhydrogen uptake despite changes to K_(L)a. This may result from theknown action of S(IV) compounds as inhibitors of microbial growth.Therefore the invention may benefit from monitoring of the level ofprovided S(IV) such that enough is provided to avoid any adverse effectof providing too little bioavailable sulfur, while avoiding the toxicityeffects of too much S(IV). Again, the addition of excessive sulfur canalso lead to the microbial culture releasing high levels of hydrogensulfide that is hazardous and expensive to remove.

The proposed method of assimilation of sulfurous acid to the cell beginswith sulfite reductase. At the working culture pH range of 4.5-5.3,sulfurous acid will chemically deprotonate to mainly form HSO₃ ⁻ as thetwo pKa's of the species are 7.2 and 1.9. This bisulfite would then bebiologically converted to H₂S via this enzyme, at which point it isbelieved to assimilate to cysteine via acetylserine coupling. While itis not known if the gene, O-acetylserine sulfhydrolase, for thisreaction is present in genome of the carboxydotrophic organisms of thisinvention, it is known that the sulfite reductase gene is in the genomeof carboxydotrophic organisms of this invention. In addition, thepresence of hydrogen sulfide in the exhaust gas of the fermenter, whilenot in the feed to the fermenter of the nutrient liquid, combined withthe ability of the microorganisms to live on this S(IV) indicate thatthe O-acetylserine sulfhydrolase gene is present.

Sulfur may be added to fermentation medium by any suitable means. Inaccordance with this invention the sulfur additive may be added in anymanner that makes the desired sulfur species biologically available tothe organism in predetermined amounts at selected times in thefermentation. Preferably, the additive is injected directly into thefermentation medium as part of nutrient liquid addition that suppliesother nutrients to the microbial culture. Thus the sulfur additive canbe added intermittently or continuously in mixtures and at aconcentration that can vary over the time of the fermentation. Thesulfur additive may be supplied to the microbial culture at differentlocations of the culture, For example, at multiple addition points in abioreactor having a fermentation vessel that suspends the microorganismsin a planktonic state. Supply of the sulfur additive will usually followinoculation of the bioreactor with microbial culture and will increaseas the culture grows and the metabolic sulfur needs of the fermentationincrease. Supply of the additive is usually regulated to maintain apredetermined concentration of one or more of the hydrosulfur or sulfuroxoanions that the microorganisms will assimilate in the bioreactor.

Any suitable method may be used for determining the concentration of thesulfur additive in solution. The supply of the sulfur additive may beregulated in response to the presence of sulfide in the off-gas from thefermentation zone. The fermentation will typically release sulfide tothe head space or gas phase above the fermentation medium. This sulfidemay originate from sulfide that formed in the fermentation medium andnever entered the microorganism (extracellular sulfide) or from sulfideexhausted from the microorganism (intracellular sulfide). This inventionadvantageously minimizes any formation of extracellular sulfide and thepresence and the release of sulfide is an indication of oversupply ofthe sulfur additive to the fermentation medium. Therefore, monitoringthe concentration of sulfide in the off-gas provides a good indicationof whether the sulfur is present in a sufficient or excessive amountrelative to the biological needs of the microorganisms. A significantpresence of sulfide in the off-gas, typically greater than 0.05 vol %indicates an excess supply of the sulfur additive. Typically a sulfideconcentration of 0.01% to 0.025% in the off-gas indicates a satisfactoryaddition rate of the sulfur additive. The absence of sulfide in theoff-gas may indicate a shortage in meeting the biological requirementsof the microorganisms and could affect the productivity of themicroorganisms over time.

Some embodiments of this invention may advantageously use cysteine ininitiating a fermentation operation. Cysteine may be beneficial in theinitial passaging up of the microbial culture in to larger volumefermentations. The relatively low volumes of fermentation medium used inthe initial growth stages of fermentation still allow the cost effectiveuse of cysteine as part of initially bringing a fermentation operationup to commercial volumes. For instance, the use of cysteine in firstseries of fermentation vessels that provide the initial passaging of theseries of reactors will only involve volumes from a few liters to a fewthousand liters and will not impose any substantial economic penalty tothe use of some cysteine. The greatest advantages to this invention comewith the discovery that cysteine may be substantially reduced oreliminated from commercial fermentation zones that have liquid volumesof one million liters or more and the use of cysteine as the primarysulfur source in such fermentations would require 100 kilograms or moreof cysteine per day.

In other embodiments the fermentation of this invention may benefit fromthe addition of certain amino acids to the fermentation medium. In thecase where cysteine is phased out of an initial fermentation it may beadvantageous to add one or supplemental amino acids to the fermentationmedium during the transition of the microbial culture from cysteine tothe sulfur additives of this invention. Amino acids such as methionineand serine may be useful in this regard, in particular serine.

In addition to the sulfur, the liquid nutrient medium will include abroad range of minerals, trace metal components, and vitamins. Tracemetals are part of the liquid nutrient medium and ordinarily willinclude one or more of manganese, zinc, molybdenum, selenium, tungsten,iron, cobalt and nickel. The composition of various nutrient media foranaerobic fermentation is well known and are described in U.S. Pat. Nos.5,173,429 and 5,593,886. Typical compounds and final concentrations forthe nutrients are given in Tables 1 to 3.

TABLE 1 Minerals Components Concentration Range (g/L) NaCl  0.5 to 4.0NH₄Cl 1.25 to 5.0 KCl 0.125 to 0.5  KH₂PO₄ 0.125 to 0.5  MgSO₄•7H₂O 0.25to 1.0 CaCl₂•2H₂O 0.05 to 0.2

TABLE 2 Trace Metals Components Concentration (mg/L) MnSO₄•H₂O 1.9 to7.6 FeSO₄•7H₂O 18.3 to 73.2 CoCl₂•6H₂O 1.8 to 7.2 ZnSO₄•7H₂O 1.0 to 4.0NiCl₂•6H₂O 0.4 to 1.6 Na₂SeO₄ 0.5 to 2.0 Na₂WO₄•2H₂O 0.6 to 2.4

TABLE 3 Vitamins Components Concentration (mg/L) Thiamine•HCl 0.05 to0.2 Calcium Pantothenate 0.05 to 0.2 Nicotinic acid 0.005 to 0.1  Biotin0.01 to 0.1

Any or all of the nutrients may be blended into one or more nutrientliquids for addition to the microbial culture. Nutrient liquids aretypically added to the fermentation medium that contacts themicroorganisms. Various blends of the nutrient liquid containingdifferent nutrients may be mixed in batch form and added to thefermentation medium on a continual or intermittent basis. In a preferredembodiment, the nutrient liquid be mixed just upstream of entering thefermentation medium by receiving serial addition of solutions containingnutrient components. The use of multiple nutrient liquid stream and/orserial addition of component solution may aid in avoiding precipitationof nutrient components.

The addition rates of the various nutrients into the microbial culturemay be determined by any suitable method. Such methods includedetermining the average needs of the microbial culture at differentstages of growth and productivity and calculating the additions rates ofthe various components at different points in time. Monitoring may alsotake place by various sampling means in which the levels of thedifferent nutrient components are directly measured and additions ratesset or adjusted in response to predetermined ranges for such nutrients.The sampling may take place by the use of probes or direct analysis ofrecovered samples. Methodologies for such analysis are well known in theart and include mass spectroscopy, inductively coupled plasma massspectroscopy, high performance liquid chromatography, ion exchangechromatography, atomic absorption spectroscopy, and/or atomic absorptionmass spectroscopy as examples.

The addition of the nutrients, in particular the trace metals, mayresult in the formation of metal precipitates in the fermentationmedium. Overall or localized concentration of the metal in thefermentation medium may result in such precipitates or precipitates mayform in the preparation of various nutrient liquid solutions. Theelimination of cysteine as a nutrient additive can exacerbate theformation of these precipitates since the addition of cysteine providesa chelate to the nutrient liquid and to the fermentation media.

The formation of the metal precipitate may be avoided by the addition ofa metal chelate to the nutrient liquid and/or the fermentation medium.Suitable metal chelates that may be incorporated into the nutrientand/or fermentation medium include ethylenediaminetetraacetic acid(EDTA), diethylenetriaminepentaacetic acid (DTPA), and nitrilotriaceticacid (NTA), and sodium citrate. The metal chelate may be added at anyconcentration that will avoid the formation of the precipitate.Desirable metal chelate concentrations will vary primarily with themetals concentration in the fermentation medium or the nutrient liquid,temperature of the liquid, and pH of the liquid. Typical concentrationsof the metal chelate will be in a molar ratio range of chelate to totaltransition metals (as defined by IUPAC numbering as Group 3 to 12 of thePeriodic Table) of from 0.1:1 to 5:1, preferably from 0.1 to 1.0 andmore preferably in a range of from 0.2 to 0.5.

In some cases it is desirable to control the redox potential of themicrobial culture. Such controls include altering the redox potential(ORP) of the microbial culture by altering the K_(L)a mass transfercoefficient. At other times reducing agents may be added directly to theculture on a continuous or periodic basis by injection directly into theculture, a fermentation medium, or addition to the nutrient liquid. Inparticular the sulfites and bisulfites are reducing agents such thattheir addition will serve to lower the redox potential of thefermentation medium. The redox potential is typically controlled to alevel of less than −200 mV and preferably in a range of from −300 mV to−500 mV and more preferably in a range of from −350 to −450 mV. Redoxpotential is readily measured by well-known means such as an ORP probe.It is also known that lowering of the ORP can promote preferentialproduction of alcohols over acids by the reduction of the acids.

Suitable reducing agents, in particular metal reducing agents forlowering the ORP are well known to those skilled in the art. Suitablereducing agents include Cr(II) and Ti(IV)

Preferably, the pH is kept relatively constant while adjusting the ORP.A pH of 5.3 may provide a suitable operation. The preferred pH for themicrobial culture is in a range of from 5.1 to 4.3. A pH of below 5.1 ismore typically used. For a variety of reasons a pH of 4.9 or below hasbeen found beneficial with a pH range of 4.3 to 4.9 being particularlypreferred. Lowering the pH can also slow the growth of the desiredethanol producing organisms therefore the pH is usually kept at or above4.3, but for suitable organisms a lower pH may be used. A lower pHinhibits the growth of invasive species of microorganisms such asbutyrigens, which produce chemicals such as butanol and butyric acid. Inaddition, the lower pH range, preferably 4.9 or below facilitates themaintenance of a low ORP in the microbial culture.

Any source of carbon monoxide and/or carbon dioxide and hydrogen mayserve as a substrate for the fermentation of this invention. Sources ofhigh carbon monoxide gas include coke oven gas, industrial waste gasesfrom petroleum refining or steel mill waste gas. The invention isparticularly suited for substrates that comprise syngas. Syngas may bederived from various sources, including, but not limited to,gasification of carbonaceous feedstock such as biomass, landfill gas,coal, natural gas, and petroleum.

The source of the syngas is not critical to the broad aspects of thisinvention. The syngas should, however, be free of components inconcentrations that would be unduly adverse to the microorganisms usedin the fermentation such as, but not limited to, hydrogen cyanide,alkenes, and alkynes and that would be adverse if present in the soughtoxygenated organic compound such as tars and aromatics where ethanol isthe sought product. Often the syngas contains 25 to 70, say, 30 to 65,mole percent carbon monoxide; 0 to 70, say, 30 to 65, mole percenthydrogen; and 1 to 20, say 3 to 15, mole percent carbon dioxideexcluding nitrogen, methane, and water vapor from the concentrationcalculations.

The syngas may undergo treatment to improve its suitability for use inthe fermentation. Such treatment may include the removal of particulatematter. It may also be beneficial to remove contaminants that can posetoxicity problems for the microorganisms and inhibit their growth orincrease their mortality. Removal of cyanide, tars and acetylenecompounds is particularly beneficial.

Contacting of the syngas with the microorganisms may take place in anytype of bioreactor. Such bioreactors may provide for continuous or batchfermentation of the substrate with the microorganisms. Multipleindividual vessels can be provided in the arrangement of the bioreactor.The arrangement of vessels may include separate vessels with at leastone for the growth phase of the microorganisms and at least anothervessel for a production phase where the product metabolites are producedat a higher rate than for the bioreactors in the growth phase.

The syngas is provided in a manner to enhance mass transfer of hydrogenand carbon monoxide to the aqueous fermentation medium for bioconversionby microorganisms to C2 oxygenates such as ethanol or acetate.Preferably, the syngas is continuously supplied.

The conditions of fermentation, including the density of microorganisms,liquid nutrient composition, and syngas residence time, are preferablysufficient to achieve the sought conversion efficiency of hydrogen andcarbon monoxide and will vary depending upon the design of thefermentation reactor and its operation. Typical fermentation conditionsthat will apply to bioreactors are well known. In planktonic-typefermentations that suspend the microorganisms in a liquid fermentationmedia these conditions will include pressure, temperature, gas flowrate, pH, ORP, agitation rate, media flow, and mean cell retention timefor continuous fermentation operations.

Fermentation medium is maintained under fermentation conditions and thesyngas is provided therein in a manner that maximizes its utilization.The pressure may be subatmospheric, atmospheric or super atmospheric,and is usually in the range of from about 90 to 1000 KPa absolute and insome instances higher pressures may be desirable for biofilmfermentation reactors. As most reactor designs, especially forcommercial scale operations, provide for a significant height of aqueousmenstruum for the fermentation, the pressure will vary within thefermentation reactor based upon the static head. Pressure conditionswill affect the performance of the fermentation and optimization of suchconditions depend, among other things, upon the specific microorganismsthat are used, the composition of the substrate and the type ofbioreactor. Higher pressure conditions can increase the rate of transferof substrate to an aqueous phase which will decrease the retention timeof the gas phase components in a given liquid volume to achieve adesired conversion of substrate. It has been reported in WO 02/08438that increased ethanol productivities of 10 to 20 times as many gramsper liter per day may be obtained by operating under higher pressure onthe order of 2 to 5 atmospheres. However, decreased retention times andhigher productivities must be balanced against the structuralrequirements for operating large fermentation vessel under highpressure. For this reason it may be most advantageous to operate largebioreactors at or close to atmospheric pressure. Higher pressures may beadvantageously used in bioreactors that fix microorganisms on varioussupports and therefore have relatively lower containment volumes thatmake use of higher pressures practical.

CO partial pressure can also be a limiting condition in the fermentationand a condition that will have greater impact at higher operatingpressures in the fermentation. The process needs sufficient substrate toensure continued growth and productivity by the microorganisms, howeverwhen the partial pressure of CO becomes too high it can become toxic tothe microorganisms and inhibit their growth and continued survival.

One or more carboxydotrophic microorganisms may be used in thefermentation to produce the sought oxygenated organic compound. Inparticular embodiments, the fermentation reaction is carried out by oneof more strains of acetogenic bacteria.

Bioconversions of CO and H₂/CO₂ to ethanol and acetate are well known.For example, in a recent book concise description of biochemicalpathways and energetics of such bioconversions have been summarized byA. Das and L. G. Ljungdahl, Electron Transport System in Acetogens andby H. L. Drake and K. Kusel, Diverse Physiologic Potential of Acetogens,appearing respectively as Chapters 14 and 13 of Biochemistry andPhysiology of Anaerobic Bacteria, L. G. Ljungdahl eds, Springer (2003).Any suitable microorganisms that have the ability to convert the syngascomponents: CO, H₂, CO₂ individually or in combination with each otheror with other components that are typically present in syngas may beutilized. Suitable microorganisms and/or growth conditions may includethose disclosed in U.S. patent application Ser. No. 11/441,392, filedMay 25, 2006, entitled “Indirect Or Direct Fermentation of Biomass toFuel Alcohol,” which discloses a biologically pure culture of themicroorganism Clostridium carboxidivorans having all of the identifyingcharacteristics of ATCC no. BAA-624; U.S. Pat. No. 7,704,723 entitled“Isolation and Characterization of Novel Clostridial Species,” whichdiscloses a biologically pure culture of the microorganism Clostridiumragsdalei having all of the identifying characteristics of ATCC No.BAA-622; both of which are incorporated herein by reference in theirentirety. Clostridium carboxidivorans may be used, for example, toferment syngas to ethanol and n-butanol. Clostridium ragsdalei may beused, for example, to ferment syngas to ethanol.

Suitable microorganisms and growth conditions include the anaerobicbacteria Butyribacterium methylotrophicum, having the identifyingcharacteristics of ATCC 33266 which can be adapted to and utilize COenabling the production of n-butanol as well as butyric acid as taughtin the references: “Evidence for Production of n-Butanol from CarbonMonoxide by Butyribacterium methylotrophicum,” Journal of Fermentationand Bioengineering, vol. 72, 1991, p. 58-60; “Production of butanol andethanol from synthesis gas via fermentation,” FUEL, vol. 70, May 1991,p. 615-619. Other suitable microorganisms include: Clostridiumljungdahlii, with strains having the identifying characteristics of ATCC49587 (U.S. Pat. No. 5,173,429) and ATCC 55988 and 55989 (U.S. Pat. No.6,136,577) that will enable the production of ethanol as well as aceticacid; Clostridium autoethanogenum-sp. nov., An anaerobic bacterium thatproduces ethanol from carbon monoxide, Jamal Abrini, Henry Naveau,Edomond-Jacques Nyns, Arch Microbiol., 1994, 345-351, Archives ofMicrobiology 1994, 161: 345-351; and Clostridium coskatii having theidentifying characteristics of ATCC No. PTA-10522 and issued as U.S.Pat. No. 8,143,037. All of these references are incorporated herein intheir entirety.

Suitable microorganisms for bioconversion of syngas to C2 oxygenatessuch as ethanol and acetate in accordance with this invention live andgrow under anaerobic conditions, meaning that dissolved oxygen isessentially absent from the fermentation zone. Where large quantities ofnutrient liquid or fermentation medium make-up are added to themicrobial culture, such as continuous fermentations operating with a lowmean cell retention time, the fermentation may benefit from the additionof one or more reducing agents to maintain anaerobic conditions and theORP in a predetermined range.

Other additives to the fermentation medium may comprise bufferingagents, trace metals, vitamins, minerals, salts, etc. Adjustments in thefermentation medium may create different conditions at different timessuch as growth and non-growth conditions which will affect theproductivity of the microorganisms. U.S. Pat. No. 7,704,723, herebyincorporated by reference in its entirety, discloses the conditions andcontents of suitable fermentation medium for bioconversion CO and H₂/CO₂using anaerobic microorganisms.

Anaerobic fermentation conditions include a suitable temperature, say,between 25° and 60° C., frequently in the range of about 30° to 40° C.Highly preferred temperature conditions for this fermentation includetemperatures in the range of 36° to 38° C.

The fermentation may be carried out in any of the bioreactors aspreviously described. Preferred reactors are those that can provide ahigh productivity of C2 oxygenates under industrially practicalconditions. Bubble column bioreactors are favored for their ability toconserve energy requirements for the circulation and mixing of thesubstrate in the liquid phase. Membrane supported bioreactors canprovide significant advantages in commercial operations by facilitatingoperations at higher pressure and reducing the water requirements forthe fermentation.

Product recovery typically takes place by separating desired productsfrom the fermentation medium. Any type of product recovery that recoversthe desired products from the microbial culture can be used. In mostcases the products are recovered from the fermentation medium whichcaptures the metabolites from the microorganisms by intimate contacttherewith. Usually a portion of the fermentation medium is withdrawnfrom time to time or continuously from the bioreactor for such productrecovery. In the case of fermentation that suspends the microorganismsin a planktonic state or on a carrier material the withdrawal of thefermentation medium is typically made at a point at the upper portion ofthe liquid surface in the vessel. For bioreactor arrangements that fixthe microorganisms to a solid support, such as membrane supportedbioreactors or trickle bed bioreactors, the fermentation mediumcomprises a circulating fluid of which a portion is withdrawn.

Product recovery from the fermentation medium can consist of any knownequipment arrangements for removal of residual cell material, separationand recovery of liquid products from the fermentation liquid, return ofrecovered fermentation liquid and purging of waste streams andmaterials. Either ethanol or acetate may be recovered from thefermentation liquid by methods that are well-known in the art. Suitableequipment arrangement can include filters, distillation columns,membrane systems and other separation equipment. US 2009/0215139 A1shows an arrangement for a product recovery reactor that recovers anethanol product from a bioreactor, herein incorporated by reference inits entirety. Distillation methods for the recovery of ethanol fromfermentation medium results in an ethanol containing stream having anazeotropic mixture of ethanol and water (i.e., 95% ethanol and 5%water). Where an anhydrous ethanol product is desired the ethanolcontaining stream may undergo further purification by methods well-knownin the art such as molecular sieve ethanol dehydration technology.

The fermentation will usually result in an incomplete utilization of thesubstrate. As a result, it has been found useful to recover substratecomponents from the bioreactor off-gas or from the fermentation medium.In particular carbon dioxide may be removed from the bioreactor or fromthe off-gas from a bioreactor. Any suitable carbon dioxide removalprocess may be used including amine extraction, alkaline saltextractions, water absorption, membrane separation,adsorptions/desorption, and physical absorption in organic solvents. Inpreferred aspects of the invention, the off-gas after carbon dioxideremoval will contain at least about 15, say, between 15 and 50, molepercent of total hydrogen and carbon monoxide. Preferably, the carbondioxide concentration in the off-gas after carbon dioxide removal isbetween about 2 and 40, more preferably between about 5 or 10 and 20,mole percent. The off-gas after carbon dioxide removal may contain atleast about 5, and often about 10 to 20, mole percent nitrogen.

A preferred process for removal of carbon dioxide from gases is bycontacting the gas with an aqueous solution containing oxygenatedorganic compounds. This process for removing carbon dioxide from gas tobe fed to a reactor, including between sequential fermentation stages,is disclosed in U.S. Patent Publication No. 2008/0305539, filed Jul. 23,2007, herein incorporated by reference in its entirety. See also, U.S.patent application Ser. No. 12/826,991, filed Jun. 30, 2010, hereinincorporated by reference in its entirety, which discloses contacting agas stream with a mixture of water and a surface active agent underpressure to sorb carbon dioxide and phase separating the gas and liquidstream to provide a gas stream with reduced carbon dioxide concentrationto be used as feed to a reactor. U.S. Patent Publication No.2008/0305539 A1 discloses the use of membranes to remove carbon dioxidefrom a membrane supported fermentation system to prevent dilution ofconcentrations of carbon monoxide and hydrogen in a multistage system.If desired, a portion of the carbon dioxide dissolved in the liquidphase of the fermentation medium can be removed. Any convenient unitoperation for carbon dioxide removal can be used, but the preferredoperation is separation by reducing the pressure to atmospheric or lowerpressure to flash carbon dioxide gas from the liquid phase.

EXAMPLES General Procedures

A series of continuous fermentation experiments were conducted to showthe ability of various strains of microorganism to produce ethanol andacetate products in anaerobic fermentations using the sulfur additive ofthis invention. The experiments were conducted using a starting stockfermentation medium having nutrients in the ranges as given by tables4-6. This media culture served as the base liquid media for theexperiments.

TABLE 4 Mineral Components Concentration (g/L) NH₄Cl 2.5 KCl 0.25 KH₂PO₄0.5 MgSO₄•7H₂O 0.125 CaCl₂•2H₂O 0.05

TABLE 5 Trace metal Components Concentration (mg/L) MnSO₄•H₂O 3.8 to 7.5FeSO₄•7H₂O  37 to 183 CoCl₂•6H₂O 3.6 to 7.2 ZnSO₄•7H₂O 2.0 to 9.8NiCl₂•6H₂O 0.8 to 1.6 Na₂SeO₄ 1.0 to 2.0 Na₂WO₄ 0.6 to 1.2

TABLE 6 Vitamin Components Concentration (mg/L) Thiamine•HCl 0.1 to 0.2Calcium Pantothenate 0.1 to 0.3 Nicotinic acid  0.005 to 0.0015 Biotin0.05 to 0.1 

Quantities of the base liquid media described above were prepared byintroducing 10 liters of water into 10 liter media feed carboys andadding the chemical components, with the exception of iron compounds,into the base liquid media in quantities that would provide the listednutrients within the ranges set forth above. The media feed carboys weresparged for at least one day with nitrogen gas prior to addition of ironcompounds and any other compounds to remove dissolved oxygen.

The procedures to establish the fermentation runs in the differentexperiments were conducted in the same manner unless otherwise noted.The standard procedure for the experiments started with the introductionof a “seed” culture in a seed vessel comprising a Biostat B supplied bySartorius Stedim Biotech, Germany. An inoculum was added to either 4.5 Lor 9 L of the liquid media in the seed vessel, depending on seed vesselvolume. Fermentation continued in the seed vessel until an opticaldensity (OD) of at least 1.5. All measurements of OD were measured by aspectrophotometer at a wavelength of 600 nm. After achieving the OD of1.5 in the seed vessel, 1 L of microbial culture from the seed vesselwas added, via an inoculation header driven by a Masterflex peristalticpump, to a 10 liter fermentor containing 9 L of the liquid mediadescribed above. Unless otherwise noted, all continuous fermentationruns were conducted using 10-L Biostat C-DCU model fermenters obtainedfrom Sartorius Stedim Biotech, Germany. Gas flow and agitation profiles,manual changes, or a combination of the two were employed to maintainhealthy and active microbial cultures, as measured by gas uptake, OD andproduct profiles. Mean cell retention time (MCRT) was adjusted daily toreflect the equation MCRT=15/OD. Liquid media from the carboy was filtersterilized upon addition to the fermenter. All of the fermentations inthe 10-L fermenters were at a super-atmospheric pressure and thepressure values represent gauge pressures unless otherwise noted. Theoff-gas from the fermenter was continually monitored using a massspectrometer to measure the uptake of CO and hydrogen. Concentrations ofproduct metabolites in the fermenter were analyzed by gas chromatographyequipped with a flame ionization detector.

Example 1

A seed culture comprising Clostridium coskatii obtained from cryogenicstocks of the microorganism on deposit as ATCC No. PTA-10522 anddescribed in U.S. Pat. No. 8,143,037 as described in US Patent was grownin a seed vessel and then introduced into the fermentor at the beginningof the continuous fermentation run according to the General Proceduresas previously described herein. At the initiation of the run (hour 0)the base liquid media entering the fermenter contained cysteine at aconcentration of 0.6 mM which provided cysteine in an amount equal toabout 0.5× the typical base reference concentration for the molaraddition of cysteine as the sole sulfur source to the fermenter. Inaddition, 1.2 mM of metabisulfite (average 0.2 mM/day/OD) was added tothe liquid media to provide a 2× addition of sulfur atoms along with 1.2mM of serine to provide 1× addition of an amino acid constituent. DTPAwas used as a chelant in this run at a concentration of 0.79 g/L.Throughout the run gas flow remained relatively constant and ranged fromabout 100 to 300 ml/min and had a volumetric composition of 34% CO, 8%CO₂, 43% H₂ and 13% CH₄. Base liquid media addition was varied within arange of about 1.6 to 3.0 liters per day. Pressure throughout the runwas maintained at 910 mbar and temperature throughout the run remainedat approximately 37° C. At hour 185 of the run a base liquid media thatno longer contained cysteine was supplied from the 10 L carboy. At hour688 the serine concentration was dropped to 0.5× and then eventually to0 at hour 808. The pH in the fermenter was initially maintained at valueof about 5.3 and was allowed to fall starting at hour 185 until itreached a value of about 4.5 around hour 288. It was brought back up to4.8 at about hour 330 and ranged between 4.8 and 4.5 through about hour800.

FIG. 1 shows the operational values for the phase of the run startingfrom hour 0 through 900 hours. Agitation varied from about 290 to 475rpm for providing some adjustment of the K_(L)a to improve stabilizationof the gas uptake. FIG. 1 shows a relatively stable uptake of CO throughthis phase of the run and that good utilization of the CO continued inthe absence of cysteine. The run phase also showed overall goodutilization of the hydrogen by its high uptake over most hours of thefermentation. Once serine was completely removed at 808 hours theremaining phases of the run showed a decline of the CO and hydrogenuptake. Colorimetric analysis of the off-gas also showed from 0 to 1350ppm sulfide. After hour 808 the run became unstable and the run wasterminated after unsuccessful attempts to stabilize it.

FIG. 2 shows a plot of the recovery of products for the 0 to 900 hourrun phase. Overall the results of the product analysis show a highconcentration of ethanol in the fermenter throughout the run phasedespite the elimination of cysteine from the liquid media feed. Theproduct data also shows a relatively low productivity of free aceticacid and total acetate. Throughout the run phase butanol and butyrateproduction were below detection limit of the gas chromatograph (˜0.1g/l).

Example 2

A seed culture comprising Clostridium coskatii obtained from the samecryostock as described in Example 1 was introduced into the fermentor atthe beginning of the continuous fermentation run. At the initiation ofthe run the liquid media entering the fermenter contained cysteine at aconcentration of 0.96 mM which provided a 0.8× normalized molar additionof sulfur to the fermenter (˜0.064 mM/day/OD). Gas flow to the runstarted at about 100 ml/min, was increased step wise until reaching avalue of about 200 ml/min at about hour 200 and remained at about 190 to200 ml/min for the rest of the run. The gas had a volume percentcomposition of 34% CO, 10% CO₂, 43% H₂ and 12% CH₄.

Base liquid media addition was varied within a range of about 1.7 to 2.9liters per day. DTPA was used as a chelant in this run at aconcentration of 0.79 g/L. Pressure throughout the run was maintained at910 mbar and temperature throughout the run remained at approximately37° C. The pH of the fermentation medium was at approximately 5.3throughout the run. After about 250 hours thiosulfate was added to theliquid media at a concentration of 1.9 mM to provide a 1.6× addition ofthiosulfate as a bioavailable sulfur addition. In addition, the liquidmedia was also amended at the same time with 1.2 mM (˜0.8 mM/day/OD) ofL-serine to provide 1× of an amino acid constituent and L-methionine ata concentration of 0.02 mM/day/OD to provide 0.25× of another amino acidconstituent. These conditions were established prior to the removal ofcysteine at about hour 260 to acclimate the cultures to the new sulfursource. The run continued for about 200 hours with a decrease inhydrogen consumption. Cysteine was introduced again at about hour 457 at0.6 mM for a 0.5× concentration (0.48 mM/day/OD) and thiosulfateaddition was stopped. Hydrogen consumption continued to decrease untilreaching a value of about 18 mmol/hr at about hour 540 and then rapidlyrose again to about 160 mmol/hr at about hour 600. CO uptake reached amaximum value of about 200 mmol/hr at about hour 260 and then decreasedto a range of about 170 to 140 mmol/hr for the remainder of the run.

The metabolite products in the fermentation medium were analyzed. Theethanol concentration increased in a generally linear manner untilreaching a peak concentration of between 16 to 18 grams/liter frombetween hours 250 to 430 and subsequently dropped to about 11 gram/literat about hour 575. Total acetate stayed at concentrations between 6 to 8grams/liter from about hours 90 to 275 before dropping below 2gram/liter from about hour 390 and for the rest of the run. The productdata also shows a relatively low productivity of free acetic acid, below2 grams/liter over the course of the run. Throughout the fermentationrun butanol and butyrate production were below detection limit of thegas chromatograph (˜0.1 g/l). Colorimetric analysis of the off-gas alsoshowed sulfide at a concentration ranging from 20 to 360 ppm.

Example 3

A new run was established by carrying over a batch of the culture fromthe end of the run described in Example 2 and initiating a new run forthis example. At the initiation of this run (hour 0) the base liquidmedia entering the fermenter contained cysteine at a concentration of0.6 mM (˜0.03 mM/day/OD) which provided 0.5× the typical base referenceconcentration for the standard molar addition of cysteine. In addition,2.4 mM of metabisulfite was added to the liquid media at the initiationof the run to provide a 4× addition of sulfur atoms. Also at theinitiation of the run L-methionine was added to the base liquid media ata concentration of 0.30 mM to provide a 0.25× addition of a sulfurbearing amino acid and serine was added to the base media at an averageconcentration of 1.2 mM (˜0.066 mM/day/OD) to provide a 1× addition ofnon-sulfur bearing amino acid. Diethylenetriaminepentaacetic acid Achelant of DTPA was used in this run at concentration of 0.079 g/L.Addition of cysteine and methionine to the base liquid stopped at abouthour 185. At about hour 360 metabisulfite addition to the base liquidwas changed to 1.2 mM to provide a 2× addition of sulfur. Starting atabout hour 670 sulfurous acid was added to the base liquid media at aconcentration of 0.6 mM (˜0.04 mM/day/OD) to provide a 0.5× addition ofsulfur through sulfurous acid and metabisulfite addition was decreasedfrom 1.2 mM to 0.6 mM to provide a 1× addition of sulfur frommetabisulfite. The addition of metabisulfite to the base liquid stoppedat about hour 800 at which time the addition of sulfurous acid wasincreased to 1.2 mM to provide a 1× addition of sulfur through sulfurousacid. The addition of sulfurous acid was lowered to an average of 0.6 mM(˜0.04 mM/day/OD) at hour 1000 to provide a 0.5× total sulfur addition.Throughout the run gas flow remained relatively constant at 180 ml/minuntil hour 1080 when it was adjusted down to a low of about 130 and upto a high of about 260 ml/min to improve the stability of the run. Thegas had a volumetric composition of 34% CO, 8% CO₂, 44% H₂ and 13% CH₄.After establishment of the run base liquid media addition was variedwithin a range of about 2.5 to 3.3 liter per day. Pressure wasmaintained at 910 mbar throughout the run and temperature throughout therun remained at approximately 37° C. The pH in the fermenter wasinitially maintained at value of about 5.3 until about hour 1080 andthereafter was maintained in a range of 4.8 to 4.9

FIG. 3 shows the operational values for the phase of the run startingfrom hour 0 through 1300 hours. Agitation was kept in a narrow range offrom 405 to 480 rpm over most of the phase of the run. FIG. 3 shows avery stable uptake of CO through this phase of the run and that goodutilization of the CO continued in the absence of cysteine or any otheramino acid. The run phase also showed overall good utilization of thehydrogen by its high uptake over most hours of the fermentation.Hydrogen uptake also remained relatively stable until hour 900 when itto decline and eventually became unstable. Colorimetric analysis of theoff-gas also showed sulfide present throughout most of the run at aconcentration ranging from 60 to 2600 ppm.

FIG. 4 shows a plot of the recovery of products for the 0 to 1300 hourrun phase. Concentrations of product metabolites in the fermenter wereanalyzed by the same methods previously described. Overall the resultsof the product analysis show a high titer of ethanol in the fermenterthroughout the run with the addition of metabisulfite or sulfurous acidphase despite the elimination of cysteine. After the run stabilized,FIG. 4 shows a relatively low production of acetic acid and totalacetate for the majority of the run. Throughout the run phase butanoland butyrate production were below the detection limit of the gaschromatograph (˜0.1 g/l).

Example 4

A seed culture comprising Clostridium coskatii obtained from the samecryostock as described in Example 1 was grown in a seed vessel and thenintroduced into the fermentor at the beginning of the continuousfermentation run according to the General Procedures as previouslydescribed herein. At the initiation of the run (hour 0) the base liquidmedia entering the fermenter contained serine at an averageconcentration of 1.2 mM (˜0.14 mM/day/OD) to provide 1× of an amino acidconstituent. In addition, an average of 0.6 mM (˜0.06 mM/day/OD) ofmetabisulfite was added to the liquid media to provide a 1× addition ofsulfur atoms and the seed culture was grown on metabisulfite without theaddition of cysteine. DTPA was used as a chelant in this run at aconcentration of 0.79 g/L. Gas flow was brought up over about the first120 hours to a rate of about 140 ml/min and kept in the range of 140 to200 ml/min for the first 500 hours and then increased to a rate of about300 ml/min for the remaining phase of the run. The gas had a volumetriccomposition of 34% CO, 8% CO₂, 44% H₂ and 13% CH₄. Base liquid mediaaddition was varied within a range of about 1 to 3.6 liter per day.Pressure was increased over the first 100 hours and then maintained at930 mbar gauge throughout the run phase and temperature remained atapproximately 37° C. At hour 337 the serine addition rate was dropped to0.6 mM for a 0.5× addition and then to 0 at about hour 500. At hour 337the rate of metabisulfite addition was decreased to an average of 0.3 mM(0.04 mM/day/OD) to provide a 0.5× addition rate of sulfur atoms. Atabout hour 650 the rate of metabisulfite addition was increased to anaverage of 0.5 mM to provide a 0.8× addition rate of sulfur atoms. ThepH in the fermenter was maintained at value of about 4.8 throughout thisphase of the run.

FIG. 5 shows the operational values for the phase of the run startingfrom hour 0 through 760 hours. After initial ramp-up, the agitation forthe run remained relatively constant at a 400 rpm. FIG. 5 shows arelatively stable uptake of CO through this phase of the run with afirst CO uptake rate at a gas flow rate in the range of 140 to 200mL/min and an increased CO uptake rate again after 500 hours when thegas flow rate was held steady at 300 mL/min. Hydrogen uptake alsoremained relatively steady at first at lower values and then higher athigher values that coincided with the changes in the gas flow rate. Thusgood utilization of the CO and hydrogen was found in the completeabsence of cysteine addition to the liquid media. Colorimetric analysisof the off-gas also showed sulfide concentrations between 0 and 600 ppmin the effluent gas stream.

FIG. 6 shows a plot of the recovery of products for the 0 to 760 hourrun phase. Overall the results of the product analysis show a highconcentration of ethanol in the fermenter throughout the run phasedespite the elimination of cysteine addition to the liquid media feed.Ethanol concentration increased significantly at the step up on the gasflow rate despite the elimination of all serine addition. The productdata also shows a very low productivity of free acetic acid and totalacetate at the start of the run phase, but with higher concentrations atthe higher gas flow rates and the elimination of serine the acetateconcentrations increased. Throughout the run phase butanol and butyrateproduction were below 0.3 g/l.

Example 5

A seed culture comprising the PETC strain of Clostridium ljungdahliiobtained from ATCC and having ATCC Number 55383 was grown in a seedvessel and then introduced into the fermentor at the beginning of thecontinuous fermentation run according to the General Procedures aspreviously described herein. At the initiation of the run (hour 0) thebase liquid media entering the fermenter contained cysteine at anaverage concentration of 1.2 mM (˜0.4 mM/day/OD) to provide 1× of anamino acid and sulfur constituent. Metabisulfite addition to the liquidmedia began at hour 533 at an average concentration of 0.3 mM (˜0.03mM/day/OD) to provide a 0.25× addition of metabisulfite, and a 0.5×addition of sulfur atoms. DTPA was used as a chelant in this run at aconcentration of 0.79 g/L. Gas flow was brought up to 200 mL/min overabout the first 200 hours of the run phase and then adjusted between arange of about 200 to 250 mL/min over the rest of the run phase. The gashad a volumetric composition of 29-31% CO, 18-19% CO₂, 39-41% H₂ and 11%methane. After establishment of the run, base liquid media additionvaried within a range of about 2 to 2.9 liters per day. Pressure wasincreased over the first 30 hours and then maintained at 930 mbarthroughout the run phase and the temperature remained at approximately37° C. At hour 644 the cysteine addition stopped. At hour 700 theaddition rate of metabisulfite was raised to an average of 0.4 mM (˜0.04mM/day/OD) to provide a 0.35× addition. At about hour 920, the additionrate of metabisulfite was raised to an average of 0.5 mM (˜0.07mM/day/OD) to provide a 0.45× additiona and remained at the levelthroughout the rest of the run phase. The pH in the fermenter wasmaintained at 5.3 through hour 814 at then gradually decreased untilreaching 5.0 at about hour 900 and remained between 4.9 and 5.1 for therest of run.

FIG. 7 shows the operational values for the phase of the run startingfrom hour 0 through 1150 hours. After initial ramp-up, the agitation forthe run remained relatively constant in a range of between 400 and 425rpm. FIG. 7 shows a very stable uptake of CO and hydrogen afterinitiation of the run and through the first 900 hour run phase. Theuptake of CO was stable and continued for over 250 hours after alladdition of cysteine was stopped. Hydrogen uptake also remainedrelatively stable for 200 hours following removal of all cysteineaddition. Colorimetric analysis of the off-gas also showed hydrogensulfide in the range of 0 to 350 ppm.

FIG. 8 shows a plot of the recovery of products for the 1150 hours ofthe run. The results of the product analysis show that the fermentercontinued to produce a very high titer of combined ethanol and acetateproducts. It was also noted that the elimination of the cysteineappeared to improve the selectivity of the fermenter to produce ethanol.Except for a spike of free acetic acid at about hour 500, the productdata also shows a relatively low productivity of free acetic acidthroughout the run. This spike coincided with the transition fromcysteine as sulfur source to metabisulfite as sulfur source. Throughoutthe run phase butanol and butyrate production were below 0.22 g/L.

Example 6

A seed culture comprising Clostridium coskatii obtained from the samecryostock as described in Example 1 was grown in a seed vessel and thenintroduced into the fermentor at the beginning of the continuousfermentation run according to the General Procedures as previouslydescribed herein. At the initiation of the run (hour 0) the base liquidmedia entering the fermenter contained cysteine at an averageconcentration of 1.2 mM (˜0.14 mM/day/OD) to provide 1× of an amino acidand sulfur constituent. At hour 172 the cysteine addition rate waslowered to an average of 0.24 mM (˜0.02 mM/day/OD) to provide a 0.2×addition of sulfur to the media and sulfurous acid addition to theliquid media was initiated at an average concentration of 0.3 mM (˜0.03mM/day/OD) to provide a 0.25× addition of sulfur atoms. DTPA was used asa chelant in this run at a concentration of 0.79 g/L. Gas flow wasbrought up over about the first 150 hours to a rate of about 200 to 230mL/min and stayed in this range throughout the run. The gas had avolumetric composition of 29-31% CO, 18-19% CO₂, 39-41% H₂ and 11-12%CH₄. After the run was established base liquid media addition was variedwithin a range of about 2.8 to 2.9 liters per day until about hour 511when it was increased to a range of about 3.3 to 3.4 liters per day.Pressure was increased over the first 50 hours and then maintained at930 mbar throughout the run phase and temperature remained atapproximately 37° C. At hour 283 the cysteine addition rate was droppedto zero and the sulfurous acid addition was raised to an average of 0.6mM (˜0.07 mM/day/OD) for a 0.5× sulfur addition for the remainder of therun. The pH in the fermenter was maintained at value of about 5.3 untilabout hour 350, then lowered to about 5.0 until hour 450 and thereafterwas kept in a range of 4.9 to 4.6 for throughout this phase of the run.

FIG. 9 shows the operational values for the phase of the run startingfrom hours 0 through 788. After initial ramp-up, the agitation for therun remained in a range from 420 to 435 rpm. FIG. 9 shows relativelysteady uptake of CO following hour 200 when the fermentation stabilizedfrom the changes in the sulfur source and the elimination of cysteine.Once the fermentation was established hydrogen uptake remained atrelatively high level until the very end of the run. Thus goodutilization of the CO and hydrogen was found to occur after the removalof cysteine from the liquid media. Colorimetric analysis of the off-gasalso showed hydrogen sulfide in the range of 0 to 153 ppm.

FIG. 10 shows a plot of the recovery of products for the 0 to 788 hourrun phase. The results of the product analysis show that thefermentation produced a high concentration of ethanol within the first200 hours. The concentration of ethanol in the fermenter remained in thesame high range after the elimination of the cysteine from the liquidmedia and until the end of the run. The product data also shows a highconcentration of free acetic acid and total acetate at the start of therun phase that diminished as the ethanol concentration increased.Throughout the run phase butanol and butyrate production were below thedetection limit of the GC.

Example 7

A seed culture comprising Clostridium coskatii obtained from the samecryostock as described in Example 1 was grown in a seed vessel and thenintroduced into the fermentor at the beginning of the continuousfermentation run according to the General Procedures as previouslydescribed herein. At the initiation of the run (hour 0) the base liquidmedia entering the fermenter contained cysteine at an averageconcentration of 1.2 mM to provide 1× of an amino acid and sulfurconstituent. At hour 172 the cysteine addition rate was lowered to anaverage of 0.24 mM to provide a 0.2× addition of sulfur to the liquidmedia and bisulfite addition to the liquid media was initiated at anaverage concentration of about 0.4 mM (˜0.04 mM/day/OD) to provide a0.33× addition of sulfur atoms. DTPA was used in this run as a chelantfrom hour 0-818 at a concentration of 0.079 g/L. After hour 818 nochelant was used. Gas flow was brought up at this point to about 230mL/min and kept in range of from 200 to 250 mL/min until about 800 hoursafter which it was brought up to about 300 mL/min where it remained forthe rest of the phase of the run. The gas had a volumetric compositionof 29-31% CO, 18-19% CO₂, 39-41% H2 and 11-12% CH₄. After establishmentof the run the base liquid media addition was varied within a range ofabout 2.5 to 2.9 liters per day until about hour 509 when it wasincreased to a range of about 3.3 to 3.4. Pressure was increased overthe first 50 hours and then maintained at 930 mbar throughout the runphase and temperature remained at approximately 37° C. At hour 283 thecysteine addition rate was dropped to zero and the bisulfite additionwas raised to an average of 0.8 mM (˜0.1 mM/day/OD) for a 0.66×concentration until hour 988 when it was raised to about 1.6 mM (˜0.2mM/day/OD) for a 1.33× concentration for the rest of the run phase. ThepH in the fermenter was maintained at 5.3 until about hour 400, thenlowered to about 5.0 until hour 620 and thereafter was kept at 4.7 forthe remaining phase of the run.

FIG. 11 shows the operational values for the phase of the run startingfrom hour 0 through 1100 hours. After initial ramp-up, the agitation forthe run phase remained in a range of from 400 to 435 rpm. FIG. 9 showsthe fermenter reaching a high uptake of CO at about hour 200 with ageneral increase of CO uptake throughout the run phase even after theelimination of cysteine. The hydrogen uptake generally tracked the COuptake but with greater variability with both uptake measurementsdemonstrating the fermenter ability to utilize higher gas flow withoutthe continued addition of cysteine to the liquid media. After hour 1100both CO uptake and hydrogen uptake fell off sharply and the run wassubsequently terminated. Colorimetric analysis of the off-gas alsoshowed hydrogen sulfide in the range of 17 to 206 ppm. FIG. 12 shows aplot of the recovery of products for the same run phase. The results ofthe product analysis show that the fermentation produced a concentrationof ethanol within the first 200 hours. The concentration of ethanol inthe fermenter remained in the same high range after the elimination ofthe cysteine from the liquid media and until the end of the run phasewhen the run became unstable. The product data also shows a highconcentration of free acetic acid and total acetate at the start of therun phase that diminished as the ethanol concentration increased.

Example 8

A seed culture comprising Clostridium coskatii obtained from the samecryostock as described in Example 1 was grown in a seed vessel and thenintroduced into the fermentor at the beginning of the continuousfermentation run according to the General Procedures as previouslydescribed herein. At the initiation of the run (hour 0) the base liquidmedia entering the fermenter contained cysteine at an averageconcentration of 1.2 mM to provide a 1× of an amino acid and sulfurconstituent. At hour 172 the cysteine addition rate was lowered to anaverage of about 0.24 mM to provide a 0.2× addition of sulfur to theliquid media and metabisulfite addition to the liquid media wasinitiated at an average concentration of about 0.3 mM (˜0.03 mM/day/OD)to provide a 0.25× addition of sulfur atoms. DTPA was used as a chelantin this run at a concentration of 0.79 g/L from hour 0 to 796. Nochelant was used after hour 796. Gas flow was brought up at this pointto about 230 mL/min and kept in range of from 180 to 230 mL/min untilabout 800 hours after which it was brought up to about 300 mL/min whereit remained for the rest of the phase of the run. The gas had avolumetric composition of 29-31% CO, 18-19% CO₂, 39-41% H₂ and 11-12%CH₄. After the run was established base liquid media addition was variedwithin a range of about 2.5 to 2.9 liters per day for about the first500 hours and then to a range of about 3.3 to 4.0 liters per day. Baseliquid media addition was varied within a range of about 2.2 to 3.0liter per day. Pressure was increased over the first 50 hours and thenmaintained at 930 mbar throughout the run phase and temperature remainedat approximately 37° C. At hour 283 the cysteine addition rate wasdropped to zero. At hour 340 the metabisulfite addition was raised to anaverage of about 0.4 mM (˜0.06 mM/day/OD) for a 0.35× addition ofmetabisulfite and a 0.7× addition of sulfur until hour 548 when it wasraised to about 0.54 mM for a 0.45× addition of metabisulfite and 0.9×addition of sulfur for the rest of the run phase. The pH in thefermenter was maintained at value of about 5.3 until about hour 170,then raised to about 5.4 until about hour 215 when it returned to 5.3and remained there until about hour 360 after which it was graduallylowered to a range of from 4.5 to 4.7 for the remaining phase of therun.

FIG. 13 shows the operational values for the phase of the run startingfrom hour 0 through 950 hours. After initial ramp-up, the agitation forthe run phase remained in a range of from 390 to 420 rpm. FIG. 13 showsthe fermenter reaching a high uptake of CO and hydrogen at about hour173. CO and hydrogen uptake persisted throughout the run phase evenafter the elimination of cysteine. The hydrogen uptake generally trackedthe CO uptake but with greater variability with both measuresdemonstrating the fermenter ability to utilize higher gas flow withoutthe continued addition of cysteine to the liquid media. After hour 1100both CO uptake and hydrogen uptake fell off sharply and the run wassubsequently terminated. Colorimetric analysis of the off-gas alsoshowed hydrogen sulfide in the range of 18 to 200 ppm. FIG. 14 shows aplot of the recovery of products for the same run phase. The results ofthe product analysis show that the fermentation produced a concentrationof ethanol within the first 200 hours. The concentration of ethanol inthe fermenter remained in the same high range after the elimination ofthe cysteine from the liquid media and until the end of the run phasewhen the run became unstable. The product data also shows a highconcentration of free acetic acid and total acetate at the start of therun phase that diminished as the ethanol concentration increased.Throughout most of the run phase butanol and butyrate production weredetection limit of the gas chromatograph (˜0.1 g/l).

Example 9

A seed culture comprising Clostridium autoethanogenum obtained from themicroorganism collection of DSMZ and having a DSM No. 10061 was grown ina seed vessel and then introduced into the fermentor at the beginning ofthe continuous fermentation run according to the General Procedures aspreviously described herein. At the initiation of the run (hour 0) thebase liquid media entering the fermenter contained cysteine at anaverage concentration of 1.2 mM to provide 1× of an amino acid andsulfur constituent. At hour 150 the cysteine addition rate was loweredto an average of about 0.24 mM to provide a 0.2× addition of sulfur fromcysteine to the liquid media and metabisulfite addition to the liquidmedia was initiated at an average concentration of about 0.3 mM (˜0.04mM/day/OD) to provide a 0.5× addition of sulfur atoms. DTPA was used asa chelant in this run at a concentration of 0.79 g/L. At hour 144 gasflow was brought up at this point to about 220 mL/min and kept in rangeof from 220 to 240 mL/min until about 500 hours after which the runbecame unstable and was terminated. The gas had an average volumetriccomposition of 29-30% CO, 17-19% CO₂, 39-41% H₂ and 11-12% CH₄. Baseliquid media addition was varied within a range of about 2.8 to 2.9liters per day. Pressure was increased over the first 30 hours and thenmaintained at 930 mbar throughout the run phase and temperature remainedat approximately 37° C. At hour 262 the cysteine addition rate wasdropped to zero. The pH in the fermenter was maintained at a value ofabout 5.3 until about hour 431, then gradually lowered throughout therun phase to about 4.9.

FIG. 15 shows the operational values for the phase of the run startingfrom hour 0 through 900 hours. After initial ramp-up, the agitation forthe run phase remained in a range of from 415 to 425 rpm. FIG. 15 showsthe fermenter reaching its highest uptake of CO at about hour 150 with alowered but still high and stable uptake of CO throughout the rest ofthis phase of the run even after the elimination of cysteine. Thehydrogen uptake generally tracked the CO uptake but with greatervariability with both measures demonstrating the fermenter ability to ahigh gas uptake without the addition of cysteine to the liquid media.After hour 550 both CO uptake and hydrogen uptake fell off sharply andthe run was subsequently terminated. Colorimetric analysis of theoff-gas also showed hydrogen sulfide in the range of 0 to 335 ppm. FIG.16 shows a plot of the recovery of products for the same run phase. Theresults of the product analysis show that the fermentation produced aconcentration of ethanol within the first 200 hours. The concentrationof ethanol in the fermenter remained in the same high range after theelimination of the cysteine from the liquid media and until the end ofthe run phase when the run became unstable. The product data also showsa high concentration of total acetate at the start of the run phase thatdiminished as the ethanol concentration increased. Throughout most ofthe run phase butanol and butyrate production were below the detectionlimits of the GC.

Example 10

A seed culture comprising Clostridium coskatii obtained from the samecryostock as described in Example 1 was grown in a seed vessel and thenintroduced into the fermentor at the beginning of the continuousfermentation run according to the General Procedures as previouslydescribed herein except the fermentor was a Biostat B Plus and wasmaintained at 1 atm of pressure. At the initiation of the run (hour 0)the base liquid media in the vessel contained 0.12 mM bisulfite, for a0.1× sulfur addition and contained no cysteine other than cysteine thatwas present with the culture as it was transferred from the seed vessel.The media feed for the run contained bisulfite at a concentration of 0.6mM over the course of the run to provide a 0.5× of sulfur atoms. DTPAwas used as a chelant in this run at a concentration of 0.79 g/L hours 0to 318. No chelant was used after hour 318. Gas flow for this run wasrapidly brought up to 100 mL/min and at hour 242 was raised to 200mL/min and remained at the level through the first 600 hour phase of therun. The gas had an average volumetric composition of 29-30% CO, 17-18%CO₂, 40-41% H₂ and 11% CH₄. Base liquid media addition rate was kept atabout 1 liter per day until hour 313 and was then raised to 2 liters perday for the rest of the run phase. The fermenter operated at ambientpressure throughout the run. Temperature remained at approximately 37°C. The pH in the fermenter was maintained at value of about 5.3 untilabout hour 214 when it was lowered to about 4.9 for the rest of the run.

FIG. 17 shows the operational values for the phase of the run startingfrom hour 0 through 600 hours. After initiation of the run, theagitation of the fermenter was increased in stages from 375 rpm to 450rpm until hour 195, then lowered for about 20 hours to 430 and thenbegan increasing stepwise again until at about 350 hours reaching arange of from 425 to 520 where it remained for the rest of the depictedrun phase. FIG. 17 shows the fermenter with a steady increase of CO andhydrogen uptake that reached a peak at about hour 350 and then leveledoff for the remainder of the run phase. After 600 hours the run becameunstable and was terminated shortly thereafter.

FIG. 18 shows a plot of the recovery of products for the same run phase.The results of the product analysis show that the fermentation producedethanol at a steadily increasing concentration over the course of therun phase. The concentration of ethanol in the fermenter remained at itshighest level for over 300 hours. The product data shows an elevatedinitial level of total acetate that stabilized at a lower level untilthe very end of the run phase. Throughout most of the run phase butanoland butyrate production were below detection limits of the GC. Thedepicted phase of the run shows that high concentrations of C2oxygenates can be obtained with essentially only the addition ofbisulfite as the sulfur source to the fermentation.

Example 11

A seed culture comprising Clostridium coskatii obtained from the samecryostock as described in Example 1 was grown in a seed vessel and thenintroduced into the fermentor at the beginning of the continuousfermentation run that was carried out according to the procedure aspreviously described herein For example, 10. At the initiation of therun (hour 0) the base liquid media entering the fermenter contained nocysteine other than cysteine that was present with the culture as it wastransferred from the seed vessel. Bisulfite at a concentration of 0.6 mMwas supplied via the media feed over the course of the run to provide0.5× of sulfur atoms. The concentration of added bisulfite was raised toa 1× concentration for the final 40 hours of the run. DTPA was used as achelant in this run at a concentration of 0.79 g/L from hours 0 to 122.No chelant was used after hour 122. Gas flow for this run was rapidlybrought up to 100 mL/min and at hour 177 was raised to 120 mL/min overthe next 20 hours and remained at the level until increasing at hour 312to 130 mL/min for the remainder of the run. The gas had an averagevolumetric composition of 29-30% CO, 18% CO₂, 40-41% H₂ and 11% CH₄.Base liquid media addition rate was kept at about 1.25 liter per dayuntil hour 313 per day and was then raised to 1.43 liters per day forthe rest of the run. The fermenter operated at ambient pressurethroughout the run. Temperature remained at approximately 37° C. The pH,except for a short spike to 5.1 at hour 122, was kept at 4.9 throughhour 220, then lowered to 4.7 for about 40 hours, and returned to 4.8 atabout 290 hours for the remainder of the run.

FIG. 19 shows the operational values for the run starting from hour 0through 360 hours. Agitation was increased rapidly and remained at 377rpm over the first 20 hours of the run and then increased stepwise tomaximum 568 rpm at hour 232 where it remained until hour 288 after whichit was decreased to 543 at the end of the run. FIG. 19 shows thefermenter with a steady increase of CO up to about hour 200 at whichtime the uptake stabilized at high value through the end of the run.Hydrogen uptake tracked a similar increase until about hour 200 afterwhich time it fluctuated at a high level until the end of the run. FIG.20 shows a plot of the recovery of products for the same run. Theresults of the product analysis show that the fermentation producedethanol at a steadily increasing concentration over the course of therun phase up to almost 16 g/L at hour 300. The product data shows anelevated initial level of total acetate that reached its highest levelat about 100 hours and then stabilized at below 2.5 g/L through the endof the run. Throughout most of the run phase butanol and butyrateproduction were below detectable limits of the GC. The depicted phase ofthe run shows that high concentrations of C2 oxygenates can be obtainedwith essentially only the addition of bisulfite as the sulfur source tothe fermentation and at a pH well below 5.

Example 12

A seed culture comprising Clostridium coskatii obtained from the samecryostock as described in Example 1 was grown in a seed vessel and thenintroduced into the fermentor at the beginning of the continuousfermentation run according to the procedures as previously described. Atthe initiation of the run (hour 0) the base liquid media entering thefermenter contained no cysteine other than cysteine that was presentwith the culture as it was transferred from the seed vessel. Bisulfiteat a concentration of 0.6 mM was supplied via the media feed over thecourse of the run to provide 0.5× of a sulfur constituent through hour120. The concentration of added bisulfite was raised to 0.9 mM toprovide a 0.75× concentration at about 120 hours (˜0.150 mM/day/OD).Bisulfite was increased to 1.2 mM to provide a 1× concentration ofsulfur at hour 224. The chelant used in this experiment was 0.018 g/Lsodium citrate. Gas flow was initiated at about 100 mL/min and wasgradually increased to 400 mL/min at hour 488 where it remained for theremainder of the run. The gas had an average volumetric composition of29% CO, 20% CO₂, 39% H₂ and 11% CH₄. Base liquid media addition startedat about 1.25 liter per day until hour 26 and was then raised in stagesto 1.63 liters per day until hour 95, to 2.5 L/day until hour 120, to2.83 L/day until hour 153, to 3.31 L/day at hour 177 raised and then to4 liters per day for the rest of the run. The fermenter operated atambient pressure until hour 428 when it was raised to 931. Temperatureremained at approximately 37° C. The pH, started at 4.9 and wasgradually lowered to 4.5 by hour 460 and maintained at that level forthe remainder of the run.

FIG. 21 shows the operational values for the run starting from hour 0through 360 hours. Agitation began at 355 and was increased gradually to515 by hour 295 where it stayed for the remainder of the run. FIG. 19shows the fermenter with a steady increase of CO up to about hour 200 atwhich time the uptake stabilized at a high value through the end of therun. Hydrogen uptake tracked a similar increase until about hour 200after which time it fluctuated at a high level until the end of the run.

FIG. 22 shows a plot of the recovery of products for the same run. Theresults of the product analysis show that the fermentation producedethanol at a steadily increasing concentration over the course of therun phase up to almost 20 g/L at hour 700. The product data shows anelevated initial level of total acetate that reached its highest levelat about 100 hours and then stabilized at below 5 g/L through the end ofthe run. Throughout the most of the run phase butanol and butyrateproduction were at essentially zero. The depicted phase of the run showsthat high concentrations of C2 oxygenates can be obtained withessentially only the addition of bisulfite as the sulfur source to thefermentation and at a pH well below 5.

As described, the present invention provides a number of advantages,some of which have been described above and others which are inherent inthe invention. Also, modifications may be proposed without departingfrom the teachings herein. Accordingly, the scope of the invention isonly to be limited as necessitated by the accompanying claims.

It is claimed:
 1. A method for producing C2 oxygenates by anaerobicfermentation with an ethanol producing carboxydotrophic microorganism,the method comprising: (a) providing a sulfur additive comprising anS(II) to S(IV) inorganic sulfur compound that produces oxoanions ofsulfur in an aqueous fermentation medium, (b) contacting a microbialculture of the microorganism with a substrate comprising carbon monoxideand maintaining the microbial culture in the aqueous fermentation mediumcontaining the sulfur additive and having a pH of less than 5.3, and (c)recovering one or more C2 oxygenates from the aqueous fermentationmedium.
 2. The method of claim 1 wherein the oxoanions of sulfur includehydrosulfur oxoanions.
 3. The method of claim 1 wherein the sulfuradditive is an S(III) to S(IV) inorganic sulfur compound that producesoxoanions of sulfur in the aqueous fermentation medium.
 4. The method ofclaim 1 wherein the sulfur additive is selected from the groupconsisting of sulfurous acid, bisulfite, metabisulfite, dithionite,thiosulfate, and combinations thereof.
 5. The method of claim 1 whereinthe sulfur additive comprises sodium bisulfite.
 6. The method of claim 1wherein the contacting of step (b) takes place in presence of an organicsulfur source and the concentration of the organic sulfur source isreduced over a predetermined time.
 7. The method of claim 6 wherein theconcentration of added organic sulfur source in the fermentation mediumis less than 0.3 mmol organic sulfur per gram dry cell weight ofmicroorganism.
 8. The method of claim 1 wherein the microbial culture isgrown in the presence of an organic sulfur source in a first vesselprior to addition of the sulfur additive and step (b) takes place in asecond vessel separate from the first vessel.
 9. The method of claim 1wherein the sulfur additive is present in the fermentation medium in aconcentration of 0.1 to 10 mmol sulfur per gram dry cell weight ofmicroorganism.
 10. The method of claim 1 wherein the sulfur additive ispresent in the fermentation medium at a concentration between from 0.5to 2 mmol sulfur per gram dry cell weight of microorganism.
 11. Themethod of claim 1 wherein the fermentation medium is maintained with acysteine concentration of less than 0.3 mmol per gram dry cell weight ofmicroorganism.
 12. The method of claim 1 wherein the pH of thefermentation medium is in a range of 4.3 to 5.1.
 13. The method of claim1 wherein the pH of the fermentation medium is below 4.9.
 14. The methodof claim 1 wherein the fermentation medium in step (b) contains achelating agent in a molar ratio range of chelate to total transitionmetals of from 0.1 to 1.0.
 15. The method of claim 14 wherein thechelating agent is selected from the group consisting ofethylenediaminetetraacetic acid, diethylenetriaminepentaaceteic acid,nitrotriacetic acid, sodium citrate, and mixtures thereof.
 16. Themethod of claim 1 wherein the C2 oxygenate comprises ethanol.
 17. Themethod of claim 16 wherein ratio of ethanol to acetate in thefermentation medium of step (c) is such that there are at least fiveparts of ethanol for every part of acetate.
 18. The method of claim 1wherein the microorganism comprises Clostridium coskatii, Clostridiumljungdahlii, or Clostridium autoethanogenum.
 19. A method for reducingcysteine in the production of ethanol or acetate by anaerobicfermentation comprising: a) contacting a microbial culture of acarboxydotrophic microorganism with a substrate comprising carbonmonoxide and growing the microbial culture under anaerobic conditions ina first fermentation zone containing cysteine at a first concentrationof cysteine to produce a cysteine grown microbial culture in a firstfermentation zone; b) adding the cysteine grown microbial culture to asecond fermentation zone containing a sulfur additive comprising aninorganic sulfur compound having a +2 to a +4 oxidation state thatproduces anions in the second fermentation zone consisting of sulfuratoms combined with oxygen and/or hydrogen atoms and having a lowerconcentration of cysteine than the first fermentation zone; c) adding asubstrate comprising (i) carbon monoxide, (ii) carbon dioxide andhydrogen, or (iii) mixtures of (i) and (ii) to the second fermentationzone to convert the substrate to C2 oxygenates; and, d) recovering oneor more C2 oxygenates from the second fermentation zone.
 20. The methodof claim 19 wherein the concentration of cysteine in the secondfermentation zone is less than 0.3 mmol organic sulfur per gram dry cellweight of microorganism.
 21. The method of claim 19 wherein the sulfuradditive is selected from the group consisting of sulfurous acid,bisulfite, metabisulfite, and combinations thereof.
 22. The method ofclaim 19 wherein the second fermentation zone has a sulfur additiveconcentration in a range of 0.5 to 2 mmol sulfur per gram dry cellweight of microorganism.
 23. The method of claim 19 wherein a cellculture is withdrawn from the second fermentation zone and transferredto a third fermentation zone that contains the sulfur additive and areduced concentration of cysteine relative to the second fermentationzone.
 24. The method of claim 19 wherein a chelating agent is added tothe second fermentation zone.
 25. The method of claim 24 wherein thechelating agent is selected from the group consisting ofethylenediaminetetraacetic acid, diethylenetriaminepentaaceteic acid,nitrotriacetic acid, sodium citrate, and mixtures thereof.
 26. Themethod of claim 19 wherein the microorganism comprises Clostridiumcoskatii, Clostridium ljungdahlii, or Clostridium autoethanogenum. 27.The method of claim 19 wherein the first fermentation zone has a volumeof less than 40,000 liters.